Chromatography Forum

Is there an effective way to convert an HPLC system into an Flow Injection Analysis (FIA) system or simply not use a column with the HPLC instrumentation for assay (e.g. just using a backpressure regulator so that the pump works efficiently) or using an column filter instead (to mix the injection?). I'm not concerned with separating sample constituents, and, in fact, I would prefer if they were not separated. I could get away with using a standard UV/Vis instrument, but I want to utilize the HPLC autosampler in conjunction with the UV detector on the instrument.

A length of PEEK tubing in place of a column will supply enough back pressure for the pump to function, and if you had this long enough I guess it could serve as your reaction coil. My experience with FIA is you usually have a carrier and at least one reagent line. The HPLC autosampler will give you an injection into the carrier, but not sure how you plan to introduce a reagent stream.

It's the first time I've even heard about FIA. Is reagent stream constant or is it just a pulse in which the sample is introduced? In this case - can't you use binary pump and use gradient as a method of introducing reagent? Or - some autosamplers have option to create injector program ie. "take 'x' uL from vial 1, 'y' uL from vial 2, 'z' uL from vial 3, (mix 'n' times, wait for 'i' minutes etc) and inject".

I have done this using the pump to introduce the dilute reagent/carrier and injecting analyte
into this stream.

Thanks for you help. Just to clarify, I am using the term FIA in an incorrect manner here since I do not need to have a reagent stream, only a way to determine concentration by UV absorbance without any separation of sample constituents and without reaction. I will give it a shot though

So in a way you're using it as an automated UV method? Should work fine with a length of PEEK tubing for back pressure. Sorry, I thought you were adapting an FIA method, and my experience there was you have a colourimetric reagent being mixed with a carrier stream, and the reaction coil to give sufficient time for the colour to form.

There are different flavours of flow injection analysis. Some software (e.g. Chemstation) offers FIA where the result is a single data-file during the course of which the autosampler injects a whole series of vials successively. I find this less useful than setting up a sequence as though I were doing chromatography, but doing it without a column. It's easier to process the results.

Personally I prefer to use a back-pressure regulator than a long piece of tube. Some regulators are astronomically expensive, but Alltech used to do a fairly basic one for a reasonable price, so it's probably still in the Grace catalogue.

So far the capillary tubing works better than the pressure regulator as peak tailing was significantly reduced. The only issue I am encountering now is a blank peak that shows up when I inject any sample diluent into the mobile phase stream that has the same solvent (e.g. injecting "100%" pure water using "100%" pure water mobile phase). This peak is only an issue at lower wavelengths (e.g. 210nm), but does is not responsive enough at higher wavengths (e.g. 270nm) to be an issue. The UV spectrum of this peak follows somewhat like f(X) = 1/x curve. However, I want to be able to extract at lower wavelengths. Any ideas of why this is occuring?

Air?

Air/gas was my first indication as well. However, when I bypassed the degasser so that the sample diluent would have the same gas content as the mobile phase, there was still a large disturbance. I am looking into temperature effects now.

I'll take that back. The degassing affect appears to have a huge affect with solvents other than water (where there is a small perhaps negligle disturbance anyway).

Back in my early working life I was in a Hospital Lab that used low pressure FIA systems for a lot of the analyses. These low pressure systems introduced air bubbles into the tubing which prevented sample band spread as the liquid moved through the system. The bubbles were removed prior to the flow stream entering into the detector.

if you use a long length of tubing to provide the back pressure for the pumps, peak broadening may become an issue. However, if you can insert an back pressure regulator between the pump and the injector and use a zero volume union to connect the line it may be a better bet.